Particle-based immunoassay using a pegylated analyte-specific binding agent

ABSTRACT

Disclosed is a method for measurement of an analyte in a microparticle-based analyte-specific binding assay, wherein the microparticles are coated with the first partner of a binding pair, the method involving mixing the coated microparticles, an analyte-specific binding agent conjugated to the second partner of the binding pair, and a sample suspected of containing or containing the analyte, wherein the second partner of the binding pair is bound to the analyte-specific binding agent via a linker having from 12 to 30 ethylene glycol units (PEG 12 to 30), thereby binding the analyte via the conjugated analyte-specific binding agent to the coated microparticles, separating the microparticles having the analyte bound via the binding pair and the analyte-specific binding agent from the mixture and measuring the analyte bound to the microparticles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 15/899,003 filed Feb. 19, 2018, which is a continuation of International Application No. PCT/EP2016/069563 filed Aug. 18, 2016, which claims priority to EP 15181763.2 filed Aug. 20, 2015, the disclosures of which are hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

The present invention relates to a method for measurement of an analyte in a microparticle-based analyte-specific binding assay, wherein said microparticles are coated with the first partner of a binding pair, the method comprising mixing the coated microparticles, an analyte-specific binding agent bound to the second partner of the binding pair, and a sample suspected of comprising or comprising the analyte, wherein said second partner of the binding pair is bound to said analyte-specific binding agent via a linker comprising from 12 to 30 ethylene glycol units (PEG 12 to 30), thereby binding the analyte via the analyte-specific binding agent to the coated microparticles, separating the microparticles comprising the analyte bound via the binding pair and the analyte-specific binding agent from the mixture and measuring the analyte bound to the microparticles.

Numerous methods and systems have been developed for the detection and quantitation of analytes of interest in biochemical and biological samples. Methods and systems which are capable of measuring trace amounts of microorganisms, pharmaceuticals, hormones, viruses, antibodies, nucleic acids and other proteins are of great value to researchers and clinicians.

Many assay methods make use of an analyte-specific binding agent to capture a specific target molecule of interest from a sample and allow for determination of the target molecule.

A substantial body of art has been developed based upon binding reactions, e.g., antigen-antibody reactions, nucleic acid hybridization techniques, and protein-ligand systems. The high degree of specificity in many biochemical and biological binding systems has led to many assay methods and systems of value in research and diagnostics. Typically, the existence of an analyte of interest is indicated by the presence or absence of an observable “label” attached to one or more of the analyte-specific binding agents.

Assays sensitivity is largely limited by non-specific binding phenomena. Thus the main difficulty is to conceive an assay technology that is very sensitive and that at the same time does not intrinsically suffer from a high background signal, e.g. caused by the sample fluid that is probed. Non-specific binding typically leads to an increased background signal, to an inaccurate detection and to a higher (worse) detection limit. In particular, nonspecific binding is even more challenging when complex biological matrices such as human plasma or serum samples are used as sample fluids.

In recent years, more accurate and sensitive assays have been developed, which are based on the use of (e.g. superparamagnetic) microparticles. Especially in such particle-based assays, important contributions to nonspecific signals come from particle-particle interactions and/or from particle-surface interactions.

U.S. Pat. No. 5,212,063 discloses a process for the detection of analytes in body fluids containing free biotin by immunoassay which make use of biotin conjugates. The document mentions polymer microparticles consisting of a core and containing a polymer which has a plurality of binding sites for biotin and, as a covering, at least one layer of protein.

WO 2013/001447 describes a precoated microparticle having a coating comprising a shell structure, wherein said shell structure comprises a first layer comprising one or more affinity molecules (i.e. an analyte-specific binding agent) and further a second layer, which is coupled to the first layer, and wherein said first and second layer comprises non-affine spacer molecules forming a mesh, wherein said one or more affinity molecules are embedded within the coating structure and wherein said mesh generates a steric hindrance for non-specific molecules. Using such especially treated/coated microparticles the background caused by non-specific binding could be reduced.

However, even the most advanced assays still exhibit significant levels of non-specific binding impairing either the lower limit of detection (LOD) or the measuring range or both. Test developers often have to compromise between assay sensitivity, measuring range and assay specificity. At the same time the tests need to be rapid, sensitive, quantitative accurate and even cost-effective. Moreover the platform on which the test is to be performed needs to be easy to use and reliable.

There is always a desire to improve assays by increasing ratio of signal to background noise and, therefore, the sensitivity of the assay. Increasing the signal of an assay also has several instrumental advantages including: i) less sensitive (and less expensive) detection systems are required; ii) smaller amounts of valuable samples are required; iii) instrumentation may be miniaturized so as to allow for instruments that are smaller and/or devices that run many assays concurrently in a small area.

However, especially in particle-based assays, important contributions to nonspecific signals come from particle-particle interactions and particle-surface interactions.

There is thus a need to design novel structures and methods for improved particle-based assay methods. There is a strong need to avoid non-specific binding to and clustering of microparticles, which is a limiting factor in a huge number of detection assays.

It has now surprisingly been found that linker molecules comprising between 12 and 30 polyethylene glycol units—bound on the one hand to one member of a binding pair and on the other hand to an analyte-specific binding agent—can be used with great advantage in a microparticle-based binding assay, wherein the microparticles are coated with the other member of the binding pair.

SUMMARY OF THE INVENTION

Disclosed is a method for measurement of an analyte in a microparticle-based analyte-specific binding assay, wherein said microparticles are coated with the first partner of a binding pair, the method comprising a) mixing the coated microparticles, an analyte-specific binding agent bound to the second partner of the binding pair, and a sample suspected of comprising or comprising the analyte, wherein said second partner of the binding pair is bound to said analyte-specific binding agent via a linker comprising from 12 to 30 ethylene glycol units (PEG 12 to 30), thereby binding the analyte via the analyte-specific binding agent to the coated microparticles, b) separating the microparticles comprising the analyte bound via the binding pair and the analyte-specific binding agent from the mixture and c) measuring the analyte bound to the microparticles.

Also disclosed is a kit comprising in separate containers or in separated compartments of a single container unit at least microparticles coated with the first member of a binding pair and an analyte-specific binding agent bound to the second member of this binding pair, wherein said second member of said binding pair is bound to said analyte-specific binding agent via a linker comprising from 12 to 30 ethylene glycol units (PEG 12 to 30).

DESCRIPTION OF THE FIGURES

FIG. 1 Schematic illustration of bead aggregation caused by an analyte-specific binding agent bound to several biotin moieties (Bi) via a state-of-the-art linker and binding to streptavidin molecules (crosses on the beads) on different beads.

FIG. 2 Schematic illustration of no bead aggregation due to use of an analyte-specific binding agent bound to several biotin moieties (Bi) via a linker comprising between 12 and 30 ethylene glycol units and binding to streptavidin molecules (crosses on the beads) on the same bead.

FIG. 3 Pattern of beads on the measuring (working) electrode of an automated Cobas® e601 analyzer (Roche Diagnostics GmbH) as obtained in an experimental HCV core antigen assay using Biotin-DDS for biotinylation: The two pictures in the upper row represent the bead distribution in a negative or control sample. The two pictures in the lower row represent the bead distribution in an HCV-positive sample. The two pictures on the left hand side represent the bead distribution with mono-biotinylated antibody. The two pictures on the right hand side represent the bead distribution with a conjugate preparation obtained by using 3.5 equivalents of biotinylation reagent per antibody in the preparation of the conjugate. As obvious from the right hand pictures the bead distribution is very uneven/disturbed, with most beads on the left part of the electrode.

FIG. 4 Pattern of beads on the measuring (working) electrode of an automated Cobas® e601 analyzer (Roche Diagnostics GmbH) as obtained in an experimental HCV core antigen assay using Biotin-PEG24-NHS for biotinylation: The two pictures in the upper row represent the bead distribution in a negative or control sample. The two pictures in the lower row represent the bead distribution in an HCV-positive sample. The two pictures on the left hand side represent the bead distribution with mono-biotinylated antibody. The two pictures on the right hand side represent the bead distribution with a conjugate preparation obtained by using 5 equivalents of biotinylation reagent per antibody in the preparation of the conjugate. Even at this high ratio of biotinylation reagent per antibody the beads show a homogeneous distribution on the electrode.

DETAILED DISCLOSURE OF THE INVENTION

In one embodiment the present disclosure relates to a method for measurement of an analyte in a microparticle-based analyte-specific binding assay, wherein said microparticles are coated with the first partner of a binding pair, the method comprising a) mixing the coated microparticles, an analyte-specific binding agent bound to the second partner of the binding pair, and a sample suspected of comprising or comprising the analyte, wherein said second partner of the binding pair is bound to said analyte-specific binding agent via a linker comprising from 12 to 30 ethylene glycol units (PEG 12 to 30), thereby binding the analyte via the analyte-specific binding agent to the coated microparticles, b) separating the microparticles comprising the analyte bound via the binding pair and the analyte-specific binding agent from the mixture and c) measuring the analyte bound to the microparticles.

Particle-based analyte-specific binding assays are widely used in e.g. certain nephelometric assays, certain latex agglutination assays and many sensitive sandwich type assays employing a broad variety of labeling or detection techniques.

A “particle” as used herein means a small, localized object to which can be ascribed a physical property such as volume, mass or average size. Microparticles may accordingly be of a symmetrical, globular, essentially globular or spherical shape, or be of an irregular, asymmetric shape or form. The size of a particle envisaged by the present invention may vary. In one embodiment used are of globular shape, e.g. microparticles with a diameter in the nanometer and micrometer range. In one embodiment the microparticles used in a method according to the present disclosure have a diameter of 50 nanometers to 20 micrometers. In a further embodiment the microparticles have a diameter of between 100 nm and 10 μm. In one embodiment the microparticles used in a method according to the present disclosure have a diameter of 200 nm to 5 μm or from 750 nm to 5 μm.

Microparticles as defined herein above may comprise or consist of any suitable material known to the person skilled in the art, e.g. they may comprise or consist of or essentially consist of inorganic or organic material. Typically, they may comprise or consist of or essentially consist of metal or an alloy of metals, or an organic material, or comprise or consist of or essentially consist of carbohydrate elements. Examples of envisaged material for microparticles include agarose, polystyrene, latex, polyvinyl alcohol, silica and ferromagnetic metals, alloys or composition materials. In one embodiment the microparticles are magnetic or ferromagnetic metals, alloys or compositions. In further embodiments, the material may have specific properties and e.g. be hydrophobic, or hydrophilic. Such microparticles typically are dispersed in aqueous solutions and retain a small negative surface charge keeping the microparticles separated and avoiding non-specific clustering.

In one embodiment of the present invention, the microparticles are paramagnetic microparticles and the separation of such particles in the measurement method according to the present disclosure is facilitated by magnetic forces. Magnetic forces are applied to pull the paramagnetic or magnetic particles out of the solution/suspension and to retain them as desired while liquid of the solution/suspension can be removed and the particles can e.g. be washed.

The microparticles used in a method according to the present invention are coated with the first member of a specific binding pair.

A “binding pair” as used herein consists of two partners binding to each other with high affinity, i.e. with one nanomolar affinity or better. Embodiments for binding pairs are for example the binding pairs consisting of receptor and ligand, hapten and anti-hapten antibody, and binding pairs based on naturally occurring high affinity binding pairs.

One example of a receptor-ligand binding pair is a pair consisting of a steroid hormone receptor and the corresponding steroid hormone.

One type of a binding pair which is suitable for the method according to the present invention is a hapten and anti-hapten antibody binding pair. A hapten is an organic molecule with a molecular weight of 100 to 2000 Dalton, preferably of 150 to 1000 Dalton. Such small molecule can be rendered immunogenic by coupling it to a carrier molecule and anti-hapten antibodies can be generated according to standard procedures. The hapten may be selected from the group comprising sterols, bile acids, sexual hormones, corticoids, cardenolides, cardenolide-glycosides, bufadienolides, steroid-sapogenines and steroid alkaloids, cardenolides and cardenolide-glycosides. Representatives of these substance classes are digoxigenin, digitoxigenin, gitoxigenin, strophanthidin, digoxin, digitoxin, ditoxin, and strophanthin. Another suitable hapten is for example fluorescein.

Examples of binding pairs based on naturally occurring high affinity binding pairs are biotin or biotin analogues such as aminobiotin, iminobiotin or desthiobiotin and avidin or streptavidin as well as the FimG and DsF binding pair. The biotin-(strept)avidin binding pair is well-known in the art. The basic principles of the FimG-DsF binding pair are e.g. described in WO2012/028697.

In one embodiment binding pairs are selected from hapten and anti-hapten antibody, biotin or biotin analogues such as aminobiotin, iminobiotin or desthiobiotin and avidin or streptavidin, FimG and DsF, and receptor and ligand.

In one embodiment binding pairs are selected from hapten and anti-hapten antibody and biotin or biotin analogues such as aminobiotin, iminobiotin or desthiobiotin/avidin or streptavidin, FimG and DsF.

In one embodiment the binding pair is biotin (or biotin analogues such as aminobiotin, iminobiotin or desthiobiotin) and avidin or streptavidin.

In one embodiment the binding pair consists of biotin and streptavidin.

In one embodiment the binding pair according to the present invention consists of a first partner of such binding pair having a molecular weight of 10 kD or more an of a second pair of such binding pair having a molecular weight of 1 kD or less. As indicated above, the first partner of a binding pair, in one embodiment having a molecular weight of 10 kD or more, is bound (coated) to the microparticles used in a method according to the present disclosure.

In one embodiment in the microparticle-based method according to the present disclosure said first partner of the binding pair is selected from avidin and/or streptavidin, and FimG, respectively, and wherein said second partner of the binding pair is selected from biotin or biotin analogues such as aminobiotin, iminobiotin or desthiobiotin and DsF, respectively.

In one embodiment in the microparticle-based method according to the present disclosure said first partner of the binding pair is avidin and/or streptavidin and wherein said second partner of the binding pair is biotin.

The microparticles used in a method according to the present invention are “coated” with the first partner of a binding pair. Such coating is performed according to state of the art procedures. The first partner of the binding pair can be bound to the surface of the particle by adsorption, by covalent binding or a combination of both methods. The microparticles optionally can be further incubated, e.g. with proteins, like bovine serum albumin, to reduce non-specific binding of other assay components. The skilled artisan is fully aware of the methods used for such optional blocking of non-specific binding. In line with the terminology used in the art, such coated and blocked microparticles also are simply referred to as coated microparticles.

The molecules of the first partner of the binding pair are present on the microparticle in close proximity providing for many nearby binding sites for the second partner of this binding pair. For most practical/routine applications the first partner of the binding pair is coated to the microparticles at saturation concentration, resulting in an optimal coating density. As the skilled artisan appreciates, the coating density, if desired could be reduced by using sub-optimal concentrations of the first partner of a binding pair. In case a sub-optimal concentration of the first partner of a binding pair would be used for coating the person skilled in the art would choose the average coating density to match the linker length used for binding the analyte-specific binding agent to the second partner of the binding pair. In general terms: The average distance between the molecules of a first partner of a binding pair on a coated microparticle will be at most twice the length of the linker used for binding the analyte-specific binding agent to the second partner of the binding pair. The distance hereby is from the center of one molecule to the center of another molecule. As an example: The average length of a polyethylene glycol unit is about 0.38 nm. Thus a linker with 12 PEG-units has about 4.5 nm in length. In order to allow the molecules of the second partner of a binding pair to bind to the first partners of said binding pair on the same microparticle the average distance between the molecules of the first partner of the binding pair on the particle would be 9 nm or less. In one embodiment the average distance of the molecules of the first partner of a binding pair is 9 nm or less. In one embodiment the average distance of the molecules of the first partner of a binding pair is 9 nm or 8 nm, respectively. In one embodiment microparticles are used which have been coated at saturation concentration of/with the first partner of a binding pair.

An “analyte” or “analyte of interest” or “target molecule” can be any molecule which can be bound by an analyte-specific binding agent. In one embodiment, an analyte within the context of the present invention is a nucleic acid (DNA or RNA) molecule, a peptide, a protein, a drug molecule, a hormone or a vitamin. In one embodiment, an analyte within the context of the present invention is a peptide, a protein, a drug molecule, a hormone or a vitamin.

Liquid samples can be used in a method for specific in vitro-detection of an analyte in a method according to the present disclosure. The sample may be known to comprise the analyte or it may be suspected of comprising the analyte. In one embodiment a sample for in vitro diagnosis used in a method according to the present disclosure is a body fluid selected from whole blood, blood serum, blood plasma, liquor, urine or saliva. In one embodiment the sample suspected of comprising or comprising the analyte is serum, plasma or liquor. In one embodiment the sample suspected of comprising or comprising the analyte is serum or plasma.

The method for measurement of an analyte according to the present disclosure makes use of an analyte-specific binding agent. The term “analyte-specific binding agent” refers to a molecule specifically binding to the analyte of interest. An analyte-specific binding agent in the sense of the present disclosure typically comprises binding or capture molecules capable of binding to an analyte (other terms analyte of interest; target molecule). In one embodiment the analyte-specific binding agent has at least an affinity of 10′ 1/mol for its corresponding target molecule, i.e. the analyte. The analyte-specific binding agent in other embodiments has an affinity of 10⁸ l/mol or even of 10⁹ l/mol for its target molecule. As the skilled artisan will appreciate the term specific is used to indicate that other biomolecules present in the sample do not significantly bind to with the binding agent specific for the analyte. In some embodiments, the level of binding to a biomolecule other than the target molecule results in a binding affinity which is only 10%, more preferably only 5% of the affinity of the target molecule or less. In one embodiment no binding affinity to other molecules than to the analyte is measurable. In one embodiment the analyte-specific binding agent will fulfill both the above minimum criteria for affinity as well as for specificity.

In one embodiment the analyte-specific binding agent is selected from the group consisting of aptamers, peptide aptamers, proteins, oligonucleotides, and molecular imprinted polymers.

An “aptamer” as used within the context of an analyte-specific binding agent may be a short nucleic acid molecule, e.g. an RNA, DNA, PNA, CNA, HNA, LNA or ANA molecule or any other suitable nucleic acid format known to the person skilled in the art, being capable of binding an analyte.

Peptide aptamers are aptamers which are able to specifically bind to (a) protein(s), polypeptide(s) or peptide(s) comprising a specific amino acid sequence. Typically, a peptide aptamer has a peptide loop, comprising for example 10 to 20 amino acids. In the context of the present disclosure a peptide aptamer may in specific embodiments be attached at one or both ends to a scaffold structure. The scaffold structure may be any molecule, preferably a protein, e.g. a protein, which has good solubility properties. Suitable scaffold molecules would be known to the person skilled in the art. Example of suitable scaffold molecules are based on bacterial protein thioredoxin-A, and FkpA- or SlyD-chaperones, respectively. The aptamer peptide loop may preferably be inserted within a reducing active site of the scaffold molecule. Alternatively, staphylococcal protein A and domains thereof and derivatives of these domains, such as protein Z or lipocalins may be used as peptide aptamers.

Nucleic acid or peptide aptamers may be generated according to any suitable method known to the person skilled in the art, e.g. via PCR or molecular synthesis approaches or yeast two-hybrid approaches.

A “peptide” as used within the context of an analyte-specific binding agent may comprise or alliteratively consist of a stretch of 2 to 49 amino acids, amino acid derivatives or a mixture thereof. The peptide may be linear, branched, circular or mixture thereof. A peptidic analyte-specific binding agent may also be attached to a scaffold structure as defined herein above.

A “polypeptide” or “protein” as used within the context of an analyte-specific binding agent may comprise or alternatively consist of a stretch of more than about 50 amino acids, amino acid derivatives or a mixture thereof. The protein may have a linear, branched, and circular form or be comprised of a mixture of these forms.

In one embodiment the analyte-specific binding agent is a polypeptide of at least 50 amino acids. Though in theory there is no upper limit to the polypeptide length of an analyte-specific binding agent in one embodiment will have at most 10.000 amino acids.

An “oligonucleotide” as used within the context of an analyte-specific binding agent may comprise or alternatively consist of a stretch of 10 to 120 nucleotides, or of 12 to 60, or of 15 to 40 nucleotides.

An oligonucleotide analyte-specific binding agent may be an RNA molecule or a DNA molecule, or a mixture of both.

The term “molecular imprinted polymer” as used herein refers to a polymer which was formed in the presence of a molecule that is extracted afterwards, leaving a complementary cavity (an imprint) behind. Typically, a molecular imprinted polymer shows a certain chemical affinity for the original molecule. A molecular imprinted polymer may be composed of any suitable polymeric unit known to the person skilled in the art. Techniques for their production include polymerization techniques such as bulk, precipitation, emulsion, suspension, dispersion, gelation, multi-step swelling polymerization and hierarchical imprinting methods.

An “antibody” as used in the context of an analyte-specific binding agent refers to an immunoglobulin molecule and to an immunologically active portion (fragment) of an immunoglobulin molecule, i. e. antibodies or antibody fragments that contain an antigen binding site that immunospecifically binds the analyte. The immunoglobulin molecules used in a method according to the present invention can be of any type (e. g., IgG, IgE, IgM, IgD, IgA and IgY), class (e. g., IgG1, IgG2, IgG3, lgG4, lgA1 and IgA2) or subclass of immunoglobulin molecules. Antibodies can be described or specified in terms of the epitope(s) or portion(s) of a polypeptide which they recognize or specifically bind. Specific epitopes and their interaction with antibodies would be known to the person skilled in the art.

The term “analyte-specific binding” as used in the context of an antibody refers to the immunospecific binding of an antibody to an epitope on the analyte. The concept of analyte-specific binding of an antibody via its epitope on an analyte is fully clear to the person skilled in the art.

The term “antibody” herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, single chain antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two different antibodies, and antibody fragments so long as they exhibit the desired biological activity. An antibody in the sense of the present disclosure may also be part of a larger fusion molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.

An “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, an antibody is purified to greater than 95% by weight of antibody, and in some embodiments, to greater than 99% as determined by SDS-PAGE under reducing or nonreducing conditions using, for example, Coomassie blue or silver stain.

Antibodies of the immunoglobulin G class usually are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V_(H)) followed by a number of constant domains. Each light chain has a variable domain at one end (V_(L)) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light-chain and heavy-chain variable domains.

The “variable region” or “variable domain” of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as “VH.” The variable domain of the light chain may be referred to as “VL.” These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions (HVRs) both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

The “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

The antibodies used in a method according to the present invention may be from any animal origin. In one embodiment the antibodies are human, murine (e. g., mouse and rat), donkey, monkey, rabbit, goat, guinea pig, camel, horse, or chicken antibodies.

Depending on the amino acid sequences of the constant domains of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al. Cellular and Mol. Immunology, 4th ed. (W. B. Saunders, Co., 2000). An antibody may be part of a larger fusion molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.

The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody in its substantially intact form, not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain an Fc region.

“Antibody fragments” comprise a portion of an intact antibody, preferably comprising the antigen-binding region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments; single-chain antibody molecules; scFv, sc(Fv)₂; diabodies; and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab′)₂ fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

The Fab fragment contains the heavy- and light-chain variable domains and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody-hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)₂ antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

“Fv” is the minimum antibody fragment which contains a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two-chain Fv species (sc(Fv)₂). It is in this configuration that the three HVRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

The term “diabodies” refers to antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (V_(H)) connected to a light-chain variable domain (V_(L)) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, EP 404097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Holliger et al., PNAS USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies. In certain embodiments, such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that a selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target-binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal-antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal-antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.

The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2^(nd) ed. 1988); Haemmerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, PNAS USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., PNAS USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and U.S. Pat. No. 5,661,016; Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (e.g., U.S. Pat. No. 4,816,567 and Morrison et al., PNAS USA 81:6851-6855 (1984)). Chimeric antibodies include PRIMATIZED® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with the antigen of interest.

“Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a HVR of the recipient are replaced by residues from a HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all, or substantially all, of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.

A “human antibody” is one which possesses an amino-acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, for example, Li et al., PNAS USA, 103:3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refers to the regions of an antibody-variable domain which are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six HVRs; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). In native antibodies, H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies. See, e.g., Xu et al. Immunity 13:37-45 (2000); Johnson and Wu in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed, naturally occurring camelid antibodies consisting of a heavy chain only are functional and stable in the absence of light chain. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993) and Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

A number of HVR delineations are in use and are encompassed herein. The HVRs that are Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody-modeling software. The “contact” HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102 H96-H101 H93-H101

HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in the VH. The variable-domain residues are numbered according to Kabat et al., supra, for each of these extended-HVR definitions.

The expression “variable-domain residue-numbering as in Kabat” or “amino-acid-position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy-chain variable domains or light-chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy-chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy-chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.

As disclosed herein, the second partner of the binding pair is bound to the analyte-specific binding agent via a linker comprising from 12 to 30 ethylene glycol units (PEG 12 to 30).

The term “linker” denotes a bifunctional or multifunctional moiety which can be used to conjugate (link) a first moiety with a second moiety or more moieties. Conjugates comprising a first and a second moiety bound to each other can be conveniently prepared using a linker having two reactive functionalities. In such conjugate the two moieties are bound “via” this linker. As obvious to the skilled artisan in such conjugate the functional moieties of the linker are present as part of a bond and not as an unreacted functional moiety.

Without wanting to be bound to the theory, it is believed that the linker comprising from 12 to 30 ethylene glycol units is key to the surprising findings disclosed herein. In the prior art on microparticle-based assays for measurement of an analyte only short PEG-containing linker molecules are seriously considered. U.S. Pat. No. 5,521,319 discloses a novel reagent that proved very useful for biotinylation of biomolecules. The length of the linker is taught to be short, i.e. up to 5 units of ethylene oxide, preferably of only 1 to 3 units of ethylene oxide and most preferred of two such units. Contrary to this teaching it has now surprisingly been found that in a microparticle-based analyte-specific binding assay long linker molecules—comprising between 12 to 30 ethylene oxide units (=PEG 12 to 30)—are advantageous if used to couple a the second member of a binding pair, e.g. a biotin, to the analyte-specific agent.

An appropriate reagent for linking or coupling biotin via a PEG-linker to a target molecule is for example a reagent according to Formula I

As will be appreciated (n) in Formula I relates to the number of ethylene glycol units. n preferably is from 12 to 30.

The methods of the invention may be constructed in a wide variety of formats. Such formats include formats known in the art such as sandwich assays and competitive binding assays (see, e.g., the following references: Nonradioactive Labeling and Detection of Molecules, Kessler, C., ed., Springer-Verlag: Berlin 1992; The Immunoassay Handbook, Wild, D., ed., Stackton Press: New York 1994; Keller, G. H. and Manak, M. M. DNA Probes, 2nd Ed., MacMillan Publishers Ltd.: London, 1993; Tietz Textbook of Clinical Chemistry 2nd Edition, Burtis et al. Ed., W. B. Saunders and Co.: Philadelphia, 1994).

In a method according to the present disclosure an analyte is measured. As the person skilled in the art will readily appreciate the measuring of the analyte bound to the microparticles is usually made by measurement of a signal carried or generated by a label on an appropriate assay component and by calculating the concentration of the analyte from a standard curve for the analyte, i.e. thereby measuring the analyte. The assay component to which a label is usually attached is either a second analyte-specific binding agent (sandwich-type assays) or the assay makes use of the competition between a labeled analyte and the analyte in the sample. Before the label is measured, the microparticles comprising part the labeled assay component are separated from the part of the labeled assay component not bound to the microparticles.

In one embodiment the methods of the present disclosure is practiced in a sandwich assay format.

A typically sandwich assay format includes mixing a microparticle coated with the first partner of a binding pair, an analyte-specific binding agent bound to the second partner of the binding pair, a sample suspected of comprising or comprising the analyte, wherein said second partner of the binding pair is bound to said analyte-specific binding agent via a linker comprising from 12 to 30 ethylene glycol units (PEG 12 to 30), and a second analyte-specific binding agent which is detectably labeled. As obvious to the skilled artisan these components are mixed and incubated for a period of time sufficient for binding the detectably labeled analyte-specific binding agent via the analyte, the analyte-specific binding agent (bound to) the second partner of the binding pair and the first partner of the binding pair to the microparticles. In one embodiment, a sandwich assay without washing step, such mixing/incubation is performed in a single reaction vessel. The sequence of adding and mixing the four ingredients (coated microparticles, sample, analyte-specific binding agent bound to the second partner of the binding pair, and detectably-labeled analyte-specific binding agent, respectively) is not critical. In one embodiment, a sandwich assay with a washing step, the adding and mixing of microparticles coated with the first member of a binding pair, sample and analyte-specific binding agent bound to the second partner of the binding pair is performed in a single reaction vessel. After this first (analyte-capturing) step the microparticles to which the analyte is now bound are washed before adding the detectably-labeled analyte-specific binding agent. The sequence of adding and mixing of the first three ingredients (coated microparticles, sample and analyte-specific binding agent bound to the second partner of the binding pair, respectively) is not critical.

In a sandwich-type assay format in one embodiment the analyte-specific binding agent bound to the second partner of the binding pair, and the detectably-labeled analyte-specific binding agent, respectively, each bind to the analyte at different and non-overlapping epitopes.

In one embodiment the methods of the present disclosure is practiced in a competitive assay format.

A typically competitive assay format includes mixing a microparticle coated with the first partner of a binding pair, an analyte-specific binding agent bound to the second partner of the binding pair, a sample suspected of comprising or comprising the analyte, wherein said second partner of the binding pair is bound to said analyte-specific binding agent via a linker comprising from 12 to 30 ethylene glycol units (PEG 12 to 30) and an amount of analyte that is detectably labeled. As obvious to the skilled artisan these components are mixed and incubated for a period of time sufficient for binding the fraction of the detectably labeled analyte that—after competition by the analyte in the sample—is able to bind to the microparticles via the analyte-specific binding agent (bound to) the second partner of the binding pair and the first partner of the binding pair coated to the microparticles.

Methods for labeling of an analyte-specific binding agent or of an analyte are well-known to the person skilled in the art and abundantly described e.g. in Haugland (2003) Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley (1992) Bioconjugate Chem. 3:2; Garman, (1997) Non-Radioactive Labeling: A Practical Approach, Academic Press, London; Means (1990) Bioconjugate Chem. 1:2; Glazer et al Chemical Modification of Proteins. Laboratory Techniques in Biochemistry and Molecular Biology (T. S. Work and E. Work, Eds.) American Elsevier Publishing Co., New York; Lundblad, R. L. and Noyes, C. M. (1984) Chemical Reagents for Protein Modification, Vols. I and II, CRC Press, New York; Pfleiderer, G. (1985) “Chemical Modification of Proteins”, Modern Methods in Protein Chemistry, H. Tschesche, Ed., Walter DeGruyter, Berlin and New York; and Wong (1991) Chemistry of Protein Conjugation and Cross-linking, CRC Press, Boca Raton, Fla.); DeLeon-Rodriguez et al, Chem. Eur. J. 10 (2004) 1149-1155; Lewis et al, Bioconjugate Chem. 12 (2001) 320-324; Li et al, Bioconjugate Chem. 13 (2002) 110-115; Mier et al Bioconjugate Chem. 16 (2005) 240-237.

The term detectably labeled encompasses labels that can be directly or indirectly detected.

Indirectly detectably labeled refers, e.g. to labeling with a hapten and to the detection of such haptenylated compound by an anti-hapten antibody carrying a directly detectable label or to the labeling with an enzyme and to the detection of such enzyme by its corresponding enzymatic activity resulting in the conversion of an appropriate dye substrate. Various enzyme-substrate labels are available or disclosed (see e.g. U.S. Pat. No. 4,275,149). The enzyme generally catalyzes a chemical alteration of a chromogenic substrate that can be measured using various techniques. For example, the enzyme may catalyze a color change in a substrate, which can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. The chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light which can be measured (using a chemiluminometer, for example) or donates energy to a fluorescent acceptor. Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRP), alkaline phosphatase (AP), (3-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to polypeptides are described in O'Sullivan et al “Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay”, in Methods in Enzym. (ed. by J. Langone & I T Van Vunakis), Academic Press, New York, 73 (1981) 147-166.

Examples of enzyme-substrate combinations (U.S. Pat. Nos. 4,275,149; 4,318,980) include, for example: Horseradish peroxidase (HRP) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor (e.g., orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethylbenzidine hydrochloride (TMB)); alkaline phosphatase (AP) with para-nitrophenyl phosphate as chromogenic substrate; and (3-D-galactosidase ((3-D-Gal) with a chromogenic substrate (e.g., p-nitro phenyl-(3-D-galactosidase) or fluorogenic substrate 4-methylumbelliferyl-(3-D-galactosidase.

Directly detectable labels either provide a detectable signal or they interact with a second label to modify the detectable signal provided by the first or second label, e.g. to give FRET (fluorescence resonance energy transfer). Labels such as fluorescent dyes and luminescent (including chemiluminescent and electrochemiluminescent) dyes (Briggs et al “Synthesis of Functionalised Fluorescent Dyes and Their Coupling to Amines and Amino Acids,” J. Chem. Soc., Perkin-Trans. 1 (1997) 1051-1058) provide a detectable signal and are generally applicable for labeling. In one embodiment detectably labeled refers to a label providing or inducible to provide a detectable signal, i.e. to a fluorescent label, to a chemiluminescent label or to an electrochemiluminescent label, respectively.

In one embodiment according to the present disclosure the microparticle-based analyte-specific binding assay makes use of a chemiluminescent or an electrochemiluminescent label and a corresponding light detection system. The light produced by the label is measured and directly or indirectly indicates the presence or quantity of the analyte.

Electrochemiluminescent (ECL) assays provide a sensitive and precise measurement of the presence and concentration of an analyte of interest. Such techniques use labels or other reactants that can be induced to luminesce when electrochemically oxidized or reduced in an appropriate chemical environment. Such electrochemiluminescence is triggered by a voltage imposed on a working electrode at a particular time and in a particular manner. The light produced by the label is measured and indicates the presence or quantity of the analyte. For a fuller description of such ECL techniques, reference is made to U.S. Pat. Nos. 5,221,605, 5,591,581, 5,597,910, PCT published application WO90/05296, PCT published application WO92/14139, PCT published application WO90/05301, PCT published application WO96/24690, PCT published application US95/03190, PCT application US97/16942, PCT published application US96/06763, PCT published application WO95/08644, PCT published application WO96/06946, PCT published application WO96/33411, PCT published application WO87/06706, PCT published application WO96/39534, PCT published application WO96/41175, PCT published application WO96/40978, PCT/US97/03653 and U.S. patent application Ser. No. 08/437,348 (U.S. Pat. No. 5,679,519). Reference is also made to a 1994 review of the analytical applications of ECL by Knight, et al. (Analyst, 1994, 119: 879-890) and the references cited therein. In one embodiment the method according to the present description is practiced using an electrochemiluminescent label.

As mentioned above electrochemiluminescence is triggered by a voltage imposed on a working electrode at a particular time and in a particular manner. What is not mentioned in detail in the prior art is the fact the distribution of microparticles on the working electrode has a major impact on the quality of an assay. The more aggregates in between microparticles are present—as a rule of thumb—the lower the quality of one or more assay features. Aggregated particles often lead to a higher co-efficient of variation in between measurements, higher background signals and/or reduced assay sensitivity.

As can be easily imagined, the use of an analyte-specific binding agent bound to the second partner of the binding pair and comprising two or more molecules of the second binding partner per molecule of the analyte-specific binding agent can easily lead to aggregation of microparticles coated with (many molecules of) the first partner of the binding pair. Therefore in the prior art many attempts have been made to produce conjugates consisting of one molecule of analyte-specific binding agent and one molecule of the second partner of the binding pair. Methods appropriate for obtaining such 1:1 conjugates are e.g. described in U.S. Pat. No. 6,391,571.

One of the most important recent approaches for site-specific protein labeling, especially site-specific mono-labeling of proteins is to incorporate bioorthogonal functionalities into these proteins at specific sites via enzymatic reactions. For a recent review on “enzymatic labeling of proteins” see M. Rashidian et al., Bioconjugate Chemistry 24 (2013) 1277-1294. The enzymes used for site-specific conjugation covered in this review include formylglycine generating enzyme, sialyltransferases, phosphopantetheinyltransferases, O-GlcNAc post-translational modification, sortagging, transglutaminase, farnesyltransferase, biotin ligase, lipoic acid ligase, and N-myristoyltransferase.

Surprisingly, as shown throughout the examples section, an analyte-specific binding agent bound to a single molecule of the second partner of the binding pair via a linker comprising from 12 to 30 ethylene glycol units (PEG 12 to 30), e.g., a monobiotinylated antibody, leads to very good assay performance both regarding co-efficient of variation as well as signal-to noise ratio.

In addition the use of an analyte-specific binding agent bound to the second partner of the binding pair, wherein said second partner of the binding pair is bound to said analyte-specific binding agent via a linker comprising from 12 to 30 ethylene glycol units (PEG 12 to 30), appears not to require mono-biotinylation or removal of conjugates of higher than 1:1 stoichiometry obtained by use of standard coupling chemistry. As expected, conjugate preparations comprising conjugates of higher than 1:1 stoichiometry tend to lead to bead aggregation. However, this effect is much less visible, pronounced if the second partner of the binding pair is bound to said analyte-specific binding agent via a linker comprising from 12 to 30 ethylene glycol units (PEG 12 to 30), even at higher than 1:1 stoichiometry.

Without wanting to be bound to this theory a possible explanation might be that these relatively long and flexible PEG linkers allow for the rapid binding of many of the second partners of the binding pair to the binding sites of the first partner of such binding pair present and within reach on the coated microparticles. To the contrary, the several second partners of a binding pair on an analyte-specific binding agent may have too short linkers to bind to another first binding partner on the same microparticle and rather tend to find an appropriate first binding partner on a second microparticle—thereby obviously promoting a tendency for bead aggregation. FIGS. 1 and 2 are attempts to schematically illustrate this theory.

In order to avoid “over-labelling” with the second partner of the binding pair the up to now standard chemistry must use a relatively low ratio of the analyte-specific binding agent and the second member of the binding pair. In order to achieve a 1:1 stoichiometry of a conjugate preparation in average usually the second partner of the binding pair (e.g. biotin in a biotinylation reagent) is used in 1.3-fold excess over the analyte-specific binding agent (e.g. an antibody). At such 1.3 to 1 coupling conditions the resulting conjugate preparation comprises about 37% of non-conjugated antibody; about 37% of mono-biotinylated antibody, but also 18%, 6% and 2% of double-, triple- or more than triple-biotinylated antibody, respectively. Usually the fraction representing the 1:1-conjugate has to be purified for achieving optimal results in a commercial immuno assay.

To the contrary, if the second partner of the binding pair is bound to said analyte-specific binding agent via a linker comprising from 12 to 30 ethylene glycol units (PEG 12 to 30), standard coupling chemistry can be used—even with the to be coupled/bound second member of the binding pair at higher molar ratios—and not requiring the isolation of the fraction comprising the 1:1 conjugates. As obvious this, in addition to the performance of such conjugates in the methods disclosed herein, is a tremendous advantage in the production of such conjugates.

In one embodiment the present disclosure relates to a conjugated specific binding agent bound to the second partner of the binding pair comprised in a composition wherein in said composition the average molar ratio between the second partner of the binding pair bound to analyte-specific binding agent is 1.1 or more.

In one embodiment the present disclosure relates to a conjugated specific binding agent bound to the second partner of the binding pair comprised in a composition wherein in said composition the average molar ratio between the second partner of the binding pair bound to analyte-specific binding agent is between 1.1 and 10.

In one embodiment the present disclosure relates to a conjugated specific binding agent bound to the second partner of the binding pair comprised in a composition wherein in said composition the average molar ratio between the second partner of the binding pair bound to analyte-specific binding agent is between 1.2 and 6.

In one embodiment the present disclosure relates to a method for measurement of an analyte in a microparticle-based analyte-specific binding assay, wherein said microparticles are coated with the first partner of a binding pair, the method comprising a) mixing the coated microparticles, an analyte-specific binding agent bound to the second partner of the binding pair, and a sample suspected of comprising or comprising the analyte, wherein said second partner of the binding pair is bound to said analyte-specific binding agent via a linker comprising from 12 to 30 ethylene glycol units (PEG 12 to 30), thereby binding the analyte via the analyte-specific binding agent to the coated microparticles and wherein said conjugated specific binding agent bound to the second partner of the binding pair is comprised in a composition wherein in said composition the average molar ratio between the second partner of the binding pair bound to analyte-specific binding agent is 1.1 or more b) separating the microparticles comprising the analyte bound via the binding pair and the analyte-specific binding agent from the mixture and c) measuring the analyte bound to the microparticles.

In one embodiment the present invention relates to a kit comprising in separate containers or in separated compartments of a single container unit at least microparticles coated with a first partner of a binding pair and an analyte-specific binding agent bound to the second partner of said binding pair, wherein said second partner of the binding pair is bound to said analyte-specific binding agent via a linker comprising from 12 to 30 ethylene glycol units (PEG 12 to 30).

The term single container unit relates to the fact that for many automatic analyzers, like the Elecsys® analyzer series from Roche diagnostics, the reagents required to measure a certain analyte are provide in the form of a “reagent pack”, i.e. as one container unit fitting on the analyzer and containing in different compartments all the key reagents required for measurement of the analyte of interest.

In one embodiment the present invention relates to a kit wherein said first partner of a binding pair is avidin or streptavidin, and wherein said second partner of said binding pair is selected from biotin or biotin analogues such as aminobiotin, iminobiotin or desthiobiotin.

In one embodiment the present disclosure relates to a kit comprising in separate containers or in separated compartments of a single container unit at least microparticles coated with avidin or streptavidin, and a biotinylated analyte-specific binding agent, wherein said biotin is bound to said analyte-specific binding agent via a linker comprising from 12 to 30 ethylene glycol units (PEG 12 to 30).

The following examples, figures and sequences are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention.

EXAMPLES

Monoclonal antibodies are prepared by standard hybridoma technology as described herein above or by recombinant NA techniques.

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The molecular biological reagents were used according to the manufacturer's instructions.

Gene and Oligonucleotide Synthesis

Desired gene segments were prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were cloned into an E. coli plasmid for propagation/amplification. The DNA sequences of subcloned gene fragments were verified by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled by annealing chemically synthesized oligonucleotides or via PCR. The respective oligonucleotides were prepared by metabion GmbH (Planegg-Martinsried, Germany).

Description of the Basic/Standard Mammalian Expression Plasmid

For the expression of a desired gene/protein (e.g. full length antibody heavy chain, full length antibody light chain, or an Fc-chain containing an oligoglycine at its N-terminus) a transcription unit comprising the following functional elements is used:

-   -   the immediate early enhancer and promoter from the human         cytomegalovirus (P-CMV) including intron A,     -   a human heavy chain immunoglobulin 5′-untranslated region         (5′UTR),     -   a murine immunoglobulin heavy chain signal sequence,     -   a gene/protein to be expressed (e.g. full length antibody heavy         chain), and     -   the bovine growth hormone polyadenylation sequence (BGH pA).

Beside the expression unit/cassette including the desired gene to be expressed the basic/standard mammalian expression plasmid contains

-   -   an origin of replication from the vector pUC18 which allows         replication of this plasmid in E. coli, and     -   a beta-lactamase gene which confers ampicillin resistance in E.         coli.

Protein Determination

The protein concentration of purified polypeptides was determined by determining the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence of the polypeptide or using the colorimetric BCA method.

Example 1 Synthesis of Activated Biotinylation Reagents

The synthesis of state-of-the-art activated biotin-comprising linkers (biotinylation reagents) like the widely used linker Biotin-DDS is disclosed in EP 632 810.

Biotin-PEGn-NHS-biotinylation reagents (CAS-Nr. 365441-71-0; n=number of ethylene oxide units) were either obtained from IRIS Biotech GmbH or synthesized in house. Biotin-PEG-110-NHS (Biotin-PEG-NHS, MW 5000 Da) was purchased from Iris Biotech GmbH (Germany).

In the de novo synthesis the control of discrete number of ethylene oxide units was ensured by the stepwise elongation of shorter PEGs, such as tetraethylene glycol, following the described method from Chen and Baker, J. Org. Chem. 1999, 64, 6840-6873.

In a first step bis-trityl-PEG_(n) 1 has been obtained (as obvious, n represents the number of ethylene glycol units).

Deprotection of 1 was carried out by stirring in 1 M HCl in dioxane for 1 h at room temperature. After evaporation the residue was refluxed in methanol until a clear solution was obtained and the flask was kept at 4° C. overnight. After filtration the solution was extracted with hexane, the methanolic layer evaporated and dried to give the corresponding PEG_(n)-diol 2 as oil or wax (consistency depending on length/number of units (n) of the PEG).

Next the introduction of the acid function was carried out by sodium catalyzed addition of PEG_(n)-diol to tert-butyl acrylate according to Seitz and Kunz, J. Org. Chem. 1997, 62, 813-826. This way compound 3 is obtained.

To a solution of HO-PEG_(n)-COOtBu 3 (1 equivalent) and triethylamine (2.5 equivalents) in methylene chloride methylsulfonyl chloride (2 equivalents) was added drop-wise at 0° C. After stirring for 1 h aqueous work-up and evaporation followed.

The mesylate 4 (1 equivalent) was directly reacted with NaN₃ (2 equivalents) by stirring in dimethylformamide at room temperature for two days. After removal of the solids and dimethylformamide, aqueous work-up with diethylether and Na₂CO₃ followed.

The crude product 5 was purified by column chromatography on silica in ethyl acetate/methanol 15/1. Reduction of the azide 5 (1 equivalent) was performed by stirring for 24 h with triphenylphosphine (1.1 equivalents) in tetrahydrofurane/water 4/1 at room temperature. After evaporation the residue was suspended in water and washed with ethyl acetate several times. The water layer was evaporated and dried to give the amine 6 as colorless oil.

Cleavage of the tert-butyl ester was carried out with 5% trifluoroacetic acid in water. Amino-PEG_(n)-acid 7 was obtained by evaporation with water for several times.

Biotin was introduced by coupling with corresponding N-hydroxysuccinimide ester 8 (1.05 equivalents) with triethylamine (4 equivalents) in dimethylformamide at room temperature overnight.

After evaporation crude product 9 was purified by RP-HPLC in acetonitrile/water.

Finally, N-hydroxysuccinimide ester 10 was formed by reaction with N-hydroxysuccinimide (1.1 equivalents) and ethyl dimethylaminopropyl carbodiimide (1.1 equivalents) in methylene chloride. After completion of the reaction, the reaction mixture was diluted with methylene chloride and washed with water. Evaporation and drying led to pure Biotin-PEG_(n)-NHS 10 as oil, wax or solid, respectively depending on number of ethylene oxide units.

Example 2

Coupling of Biotin and Ruthenium Moieties, Respectively, to Antibodies

Antibodies were obtained and purified according to state-of-the art-procedures that are fully familiar to a person skilled in the art.

For coupling, in general the lysine ε-amino groups of the antibodies were targeted by N-hydroxy-succinimide activated compounds. At a protein concentrations of 10 mg/ml antibodies were reacted with N-hydroxy-succinimide activated biotinylation reagents and N-hydroxy-succinimide activated ruthenium labeling reagents, respectively. The molar ratio of biotinylation or labeling reagent, respectively/protein (antibody) varied from 1.3:1 to 5:1, depending on the respective antibody conjugate. The reaction buffer was 50 mM potassium phosphate (pH 8.5), 150 mM KCl. The reaction was carried out at room temperature for 30 minutes and stopped by adding L-lysine to a final concentration of 10 mM. After the coupling reaction, unreacted free biotin/label was removed by passing the crude antibody conjugate through a gel filtration column (Superdex 200 HI Load) or by dialysis.

In order to obtain mono-biotinylated antibody conjugates 0.5-1 M ammonium sulfate was added to the conjugate solution. The solution was passed through a streptavidin mutein adsorber (see DE19637718) equilibrated with 50 mM potassium phosphate (pH 7.5), 150 mM KCl, 0.5-1 M ammonium sulfate. Antibodies without any biotin coupled/bound to them are unable to bind the adsorber and were washed out. Mono-biotinylated antibody conjugates were eluted with 50 mM potassium phosphate (pH 7.5), 150 mM KCl and 2% DMSO. Antibody conjugates with more than one biotin per antibody were eluted with 50 mM potassium phosphate (pH 7.5), 150 mM KCl and 2 mM biotin (“+mSA”).

Example 3

Influence of the Linker in a Biotinylation Reagent on the Immunological Detection of HCV Core Antigen

One and the same anti-HCV (capture) antibody was conjugated to biotin by use of biotinylation reagents comprising various linkers. The performance of the different biotin-linker-antibody conjugates was assessed on an automated Cobas® e601 analyzer (Roche Diagnostics GmbH).

Measurements were carried out in a sandwich assay format. Signal detection in the Cobas® e601 analyzer is based on electrochemiluminescence. In this sandwich assay the biotin-conjugate (i.e. the capture antibody) is immobilized on the surface of a streptavidin-coated magnetic bead (average bead size 2.8 μm). The detection-antibody bears a complexed ruthenium cation as the signaling moiety. In the presence of analyte, the chromogenic ruthenium complex is bridged to the solid phase and emits light at 620 nm after excitation at the platinum electrode comprised in the measuring cell of the Cobas® e601 analyzer. The signal output is in arbitrary light units. Measurements were performed with HCV core antigen positive and negative human serum and plasma samples purchased from several sources.

The experimental HCV core antigen assay was conducted as follows. 50 μl of normal or HCV antigen positive sample and 25 μl of a detergent containing pretreatment reagent to release the antigen were incubated together for 9 minutes followed by the addition of 35 μl of 2 μg/ml capture antibody-biotin conjugate and 40 μl of 1 μg/ml detection antibody ruthenium label conjugate in the same physiological buffer of pH 7.0 and comprising 100 mM potassium phosphate and 225 mM KCl. After additional 9 minutes incubation time 50 μl streptavidin-coated paramagnetic microparticles were added and incubated for further 9 minutes. Afterwards, the HCV core antigen was detected (via the electrochemiluminescent signal generated in these experiments).

TABLE 1 Comparison of different HCV capture antibody conjugates HCV capture antibody conjugate Biotin-DDS, Biotin-DDS, Biotin-PEG24, Biotin-PEG24, mono 1:3.5 mono 1:5 counts counts counts counts normal sample 1 1,247 956 1,265 960 HCV antigen 742,023 1,162,932 1,792,054 2,112,645 positive sample 1 S/N 595 1,216 1,417 2,201 normal sample 1 (21-fold measurement) mean 1,302 949 1,298 973 min 1,243 894 1,233 933 max 1,595 1,182 1,462 1,064 standard deviation 74 72 46 29 (SD) coefficient of 5.7% 7.6% 3.5% 3.0% variation (CV) HCV antigen positive sample 2 (21-fold measurement) mean 138,927 189,659 315,036 382,808 min 125,384 177,906 308,373 373,402 max 143,586 199,343 320,929 392,804 standard deviation 4,194 4,591 2,991 4,573 (SD) coefficient of 3.0% 2.4% 1.0% 1.2% variation (CV)

Generally, a high signal to noise ratio (S/N) and a low coefficient of variation (CV) particularly for background signals are important assay performance parameters especially regarding the lower limit of detection for an assay (assay sensitivity). As disclosed in EP 632 810 in streptavidin-coated microparticle based assays the Biotin-DDS biotinylation reagent is preferred over other biotinylation reagents like Biotin-NHS or Biotin-X-NHS comprising shorter or longer linkers, respectively.

In order to assess the bead distribution pattern on the working electrode the photomultiplier was replaced by a camera. In general bead aggregation of the paramagnetic streptavidin-coated microparticles and/or an inhomogeneous bead distribution pattern during the magnetic capturing on the working electrode is known to lead to partial signal loss and especially to an increased CV. It is also known that so-called matrix effects present in some samples can further increase the problems caused by bead aggregation.

The data in Table 1 show that an antibody conjugated with multiple Biotin-DDS moieties per antibody if compared to a conjugate of an antibody with only a single Biotin-DDS moiety has higher S/N values. However, this goes at the expense of a higher CV value for an analyte-free serum as compared with its single biotinylated counterpart. Furthermore, multiple biotinylation of antibodies with Biotin-DDS induces bead aggregation of the paramagnetic streptavidin-coated microparticles becoming obvious by a very inhomogeneous bead distribution pattern during the magnetic capturing on the working electrode (FIG. 3). (Since the negative serum used here is free of matrix effects, the negative impact on the CV is only moderate.) The strictly mono-biotinylated Biotin-DDS conjugate shows a much more homogeneous bead pattern on the working electrode and hence it is less prone to matrix effects (FIG. 3).

Using the Biotin-PEG24-NHS comprising a much longer linker for biotinylation to produce a mono-biotinylated conjugate the signal to noise ratio and the CV value could be increased and decreased, respectively. The bead pattern is very homogeneous, as for the mono-conjugate based on Biotin-DDS (FIG. 4).

Surprisingly, with the Biotin-PEG24 mono-conjugate very good results could be obtained, both with respect to the signal-to-noise ratio as well as regarding the co-efficient of variation. By using an antibody conjugated with multiple Biotin-PEG24-NHS biotinylation reagents per antibody a homogeneous bead distribution pattern and low CV values comparable to the results obtained for the mono-biotinylated conjugate could be obtained (FIG. 4). The signal to noise ratio rises with the increasing number of biotin residues per antibody at least in the range of biotin residues per antibody used in these experiments.

Example 4

Influence of the Linker in a Biotinylation Reagent on the Immunological Detection of Troponin T (TnT)

One and the same anti-troponin T (anti-TnT) (capture) antibody was conjugated to biotin by use of biotinylation reagents comprising various linkers. The performance of the different biotin-linker-antibody conjugates was assessed on an automated Cobas® e601 analyzer (Roche Diagnostics GmbH) based on an experimental protocol in line with the instructions for the Elecsys Troponin T hs assay kit.

Measurements were carried out in a sandwich assay format. Signal detection in the Cobas® e601 analyzer is based on electrochemiluminescence. In this sandwich assay the biotin-conjugate (i.e. the capture antibody) is immobilized on the surface of a streptavidin-coated magnetic bead (average bead size 2.8 μm). The detection-antibody bears a complexed ruthenium cation as the signaling moiety. In the presence of analyte, the chromogenic ruthenium complex is bridged to the solid phase and emits light at 620 nm after excitation at the platinum electrode comprised in the measuring cell of the Cobas® e601 analyzer. The signal output is in arbitrary light units.

Measurements were performed with human serum supplemented with defined amounts of recombinant human cardiac TnT as calibrators and results are given in Table 2.

TABLE 2 Results obtained with an experimental troponinT-assay TnT capture antibody conjugate TnT Biotin-DDS, Biotin-PEG24, Biotin-DDS, Biotin-PEG24, calibrators mono mono 1:1.3, +mSA 1:1.3, +mSA pg/ml counts counts counts counts 0 942 958 947 1,021 15 1,486 1,728 1,351 1,631 72 3,704 5,178 3,149 4,530 167 8,686 13,161 7,069 11,407 2,885 219,509 414,720 201,995 380,924 10,910 840,288 1,566,614 793,111 1,576,584

It can be concluded from the data given in Table 2, that the capture antibody conjugates produced with the long Biotin-PEG24-NHS biotinylation reagent are superior over the shorter Biotin-DDS biotinylation reagent irrespective of the average ratio between biotin and antibody. (As explained in Example 1 a conjugate with the notion “+mSA” represents the fraction comprising two or more biotin moieties per antibody.)

Example 5

Variation of Linker-Length in the Biotinylation Reagent on an HCV Core Antigen Prototype Assay

In order to determine the optimal length of the linker different Biotin-PEG(n)-NHS derivatives were conjugated with the HCV capture antibody and measured in the HCV core antigen experimental assay as described in Example 3. For each biotinylation reagent both mono-biotinylated and multiple biotinylated conjugates were generated and results are given in Table 3.

TABLE 3 Influence of PEG-linker length on performance of an HCV core antigen experimental assay HCV antigen normal positive HCV capture antibody sample 1 sample 3 conjugate counts counts S/N Biotin-PEG4, mono 1,070 380,906 356 Biotin-PEG12, mono 970 550,556 568 Biotin-PEG24, mono 884 605,566 685 Biotin-PEG30, mono 1,028 515,176 501 Biotin-PEG~110, mono 1,193 109,534 92 Biotin-PEG4, 1:1.3, +mSA 921 463,375 503 Biotin-PEG12, 1:1.3, +mSA 956 707,700 740 Biotin-PEG24, 1:1.3, +mSA 1,075 943,744 878 Biotin-PEG30, 1:1.3, +mSA 995 629,536 633 Biotin-PEG~110, 1:1.3, +mSA 1,409 697,707 495

As can be seen from Table 3, very good results as shown via the signal-to-noise ratio (S/N) have been achieved with linkers comprising between 12 and 30 ethylene glycol units. Variants generated by use of the Biotin-PEG24-NHS biotinylation reagent tended to show kind of an optimal signal to noise ratio. However, as obvious from Table 3 linkers comprising between 12 and 30 ethylene glycol units are quite superior over the shorter as well as the longer linker also tested and yield good results. 

1-12. (canceled)
 13. A kit comprising in separate containers or in separated compartments of a single container unit at least microparticles coated with a first partner of a binding pair and an analyte-specific binding agent bound to the second partner of said binding pair, wherein said second partner of the binding pair is bound to said analyte-specific binding agent via a linker comprising from 12 to 30 ethylene glycol units.
 14. The kit of claim 13, wherein said first partner of a binding pair is selected from the group consisting of avidin and streptavidin, and wherein said second partner of said binding pair is selected from the group consisting of biotin and biotin analogues.
 15. The kit of claim 13, further comprising in a separate container or in a separated compartment of a single container unit a second analyte-specific binding agent which is detectably labeled.
 16. The kit of claim 13, wherein said microparticles are from 50 nm to 20 μm in diameter.
 17. The kit of claim 13, wherein said microparticles are paramagnetic.
 18. The kit of claim 13, wherein said analyte-specific binding agent is a polypeptide of at least 50 amino acids.
 19. The kit of claim 13, wherein said analyte-specific binding agent is a polypeptide of at most 10,000 amino acids.
 20. The kit of claim 13, wherein said analyte-specific binding agent is an antibody or an antigen-binding fragment thereof. 